Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3–Expressing Liver Cancer by Immuno-PET

Abstract

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumorselective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling fulllength antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. Methods: Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with 89 Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3 1 ) tumors in vivo were determined. Results: Both 89 Zr-ssHN3 and 89 Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3 1 xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3 1 tumors. 89 Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both 18 F-FDG and 89 Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. Conclusion: 89 Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified 89 Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3directed PET imaging of liver cancers.

Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma

“…Tumor-to-liver ratios by PET/CT and biodistribution analyses were notably higher than those reported by Natarajan et al using a similar 89 Zr-labeled human αGPC3 IgG antibody and Fayn et al using 89 Zr-labeled GPC3-targeting HN3 single-domain antibodies. In addition, there was a greater relative difference between tumor uptake and uptake in organs such as the heart, lungs, gastrointestinal tract, and kidneys on biodistribution analysis [16,17]. While different methods for model development and radioimmunoconjugate injection used may affect the results such that they are not directly comparable [16,17], it is possible that our humanized antibody has a higher speci city for GPC3-expressing tumors.…”

“…In addition, there was a greater relative difference between tumor uptake and uptake in organs such as the heart, lungs, gastrointestinal tract, and kidneys on biodistribution analysis [16,17]. While different methods for model development and radioimmunoconjugate injection used may affect the results such that they are not directly comparable [16,17], it is possible that our humanized antibody has a higher speci city for GPC3-expressing tumors. Furthermore, it should be noted that tumor-to-liver ratios were measured ve days after injection in this study compared with one to seven days after injection in the aforementioned studies, however our prior experiments with 89 Zr-αGPC3 M demonstrated high tumor-to-liver ratios calculated from four hours up to seven days after injection [6,11].…”

“…While yttrium-90 microspheres and iodine-131-labeled lipiodol and metuximab are used in radioembolization therapy, there are currently no FDA approved theranostics for HCC. However, glypican-3 (GPC3)targeted radioisotopes have shown promise in preclinical and early clinical studies [6, [9][10][11][12][13][14][15][16][17][18][19][20]. GPC3 is a cell surface antigen expressed on up to 80% of HCCs but absent in liver parenchyma and benign lesions, making it an accessible and speci c target for a theranostic approach [14,15,21].…”

Imaging and Monitoring HER2 Expression in Tumors during HER2 Antibody–Drug Conjugate Therapy Utilizing a Radiolabeled Site-Specific Single-Domain Antibody Probe: 68Ga-NODAGA-SNA004-GSC

“…32−34 As expected, the omission of the His tag markedly reduced the renal uptake of 68 Ga-NODAGA-SNA004-GSC, consistent with previous studies conducted by Yan Wang et al and Xavier et al 32−34 Concurrently, the reduction in the charge of the 68 Ga/chelator complex and the creation of structurally homogeneous site-specific molecular probes notably attenuated the hepatic uptake of 68 GA-NODAGA-SNA004-GSC, leading to PET images characterized by a heightened S/N ratio. 27,30,35,36 Trastuzumab and its associated ADC therapies represent well-established modalities for managing patients afflicted with HER2-positive BC. The downregulation of HER2 facilitated by trastuzumab and its associated ADCs during therapy serves as an early prognostic indicator of treatment efficacy.…”

“…Traditional bioconjugation approaches often yield heterogeneous tracer populations and may compromise their binding affinity and pharmacokinetic properties. These limitations can be mitigated by site-specific conjugation. − Numerous studies focusing on tracers employing cysteine-based site-specificity have consistently demonstrated a higher signal-to-noise (S/N) ratio compared to those with random conjugation. , …”